The present invention relates to novel derivatized recombinant or natural proteins and/or polypeptides wherein if said protein and/or polypeptide contains one or more cysteine residue, said residue is derivatized with substituents as herein defined. Further, proteins not containing a cysteine in their natural forms can be altered, via site specific mutagenesis techniques, for instance, in order to introduce cysteines at advantageous sites and thereafter derivatize said cysteine(s).
Although molecular biological techniques have dramatically increased the availability of many proteins and/or polypeptides (hereinafter referred to as proteins), the therapeutic use of said proteins is often times hindered by other factors, such as aggregation, proteolytic degradation, poor bioavailability and physical properties which preclude efficient formulations. In the present invention the highly reactive sulfhydryl groups on cysteine residues within both recombinantly-derived and naturally-derived proteins are derivatized to modify the effective charge on the protein surface, and thereby the isoelectric point, or to link a surface-modifying polymer to the protein.
The advantage of modifying the charge iso-electric point is well known due to the minimization of protein aggregation if the pH of a protein formulation is significantly different than the iso-electric point. Manipulation of the iso-electric point, therefore, affords a technique to minimize aggregation.
Also, another mechanism for enhancing protein formulation is by conjugation of the protein with derivatizing compounds, which include, but are not limited to, polyethylene glycol and polypropylene glycol. Some of these benefits recognized include: lowered immunogenicity and antigenicity, increased duration of action, and altered pharmacokinetic properties. [F. M. Veronese, Chimicaoggi (1989) 53].
Heretofore, attachment of polymeric molecules to proteins was principally through lysine or other basic amino acids. This was accomplished with alkylating agents such as triazine derivatives, diazonium salts or active acylating agents such as succinic acid derivatives. These reactions were difficult to control and significant losses in biological activity occurred frequently due to reactions with essential amino acids in the proteins [see F. F. Davis et al Polymer (1979) 20 357].
In contrast, the present invention provides a selective method for modifying the less abundant cysteine residues either naturally occurring within a protein or incorporated within a protein by such methods as site specific mutogenesis.
It is an object of the present invention to provide compositions of derivatized proteins, which proteins include but are not limited to the (mammalian, avian and fish) somatotropins, mammalian IGF-1 and IGF-2, interleukins and interferons, prourokinase, tumor necrosis factor other growth factors such as fibroblast growth factor and antithrombin III which are biologically effective and yet stable to administer. Furthermore, as previously indicated, said proteins are not limited to those proteins which in their natural form contain cysteine residue(s) since site specific mutagenesis techniques are used to introduce cysteine at advantageous sites within said proteins. It is another object of the present invention to provide compositions of biologically active recombinant and/or natural proteins which are suitable for parenteral administration, said compositions comprising a biologically active amount of a derivatized somatotropin (animal or human form) or other protein or pharmaceutically and pharmacologically acceptable salt thereof in a pharmaceutically and pharmacologically acceptable solid or liquid carrier.
It is a further object of the present invention to provide novel compositions which will allow the modification of any cysteine-containing proteins. By judicious selection of the nature and number of ionizable-functional groups and/or the size and nature of the surface-modifying polymer a range of derivatized proteins with distinctly different physical properties are produced.
These and other objects of the invention will become more apparent by the more detailed description of the invention provided hereinbelow.